This invention relates to the fluoroassay of biological fluids. More particularly this invention relates to a substrate for fluoroimmunoassay which is capable of binding specific proteins and other biological entities (e.g. DNA). The substrate enhances the specificity and sensitivity of fluoroimmunoassay (FIA) and is thus useful in the diagnosis of diseases associated with specific antigens and antibodies. The invention is valuable in veterinary assays for diseases affecting companion, laboratory, and livestock animals as well as diseases and disease states in humans.
Fluoroassay is a powerful technique for detecting small amounts of substances in a complex mixture. It is especially useful in fluoroimmunoassay (FIA) of body fluids such as blood where small amounts of an antigen and antibodies which recognize it must be detected.
FIA is useful in diagnosis of a disease when it is specific for a particular antigen or antibody which is characteristic of that disease. However, for a reliable diagnosis, FIA must also be not only specific but also highly sensitive since the substance to be detected is often present in very small amounts in the fluid being analyzed.
Prior attempts to enhance the sensitivity of FIA include the development or use of substrates which selectively bind proteins. These substrates may be immobilized on solid surfaces so that the immunoreaction between antigen and antibody may be more easily observed.
However, these substrates have generally been comprised of polymers such as polystyrene or polypropylene which do not strongly bind proteins and thus are of limited use in selecting minute amounts of proteinaceous immunogens from complex biological fluids. A useful diagnostic assay requires a substrate which can efficiently adsorb particular antibodies or antigens.
Since FIA involves the detection of light emitted by a particular fluorophor, sensitivity also depends upon the intensity of light so emitted and its relation to background or scattered light. Choosing an incident wavelength to match excitation frequency of fluorophor is one important factor in increasing sensitivity. Harte et al. in U.S. Pat. No. 4,340,564 disclose a solid-phase immunosubstrate which comprises an over-coating of polymer beads with light scattering centers and binders which enhance scattered light. Other ways to improve substrates and increase the amount of transmitted light have been actively sought, e.g., see Pierce et al, U.S. Pat. No. 4,258,001, Mar. 24, 1981.